We have shown that this gaseous reagent, C2N2, can be used to introduce crosslinks into proteins by converting certain salt bridges and carboxyl- carboxylate pairs into covalent bonds. We intend to define (1) the types of functionalities that do react, (2) the environment of the groups that do react, and (3) for selected proteins the specific identities of residues involved. This appears to be the only protein modifying reagent specific for pairs of functional groups. A clear definition of the types of salt bridges modified by C2N2 allows the appropriate use of C2N2 to become a tool for locating salt bridge and carboxyl-carboxylate interactions. Successful development of C2N2 use as a tool, the objective of this proposal, will allow approaches to be made to long range objectives under investigation at the present time. Since the overwhelming majority of proteins remain undefined by X-ray crystallography, it will be possible (1) to extend our understanding of the importance of ion pairs, (2) develop the use of C2N2 to attach covalently ligands that are salt-bridged to the binding protein, (3) to explore the use of C2N2 to link subunits covalently that are associated in order to complete the assembly of polypeptides into the completed protein polypeptide, (4) to test for transient salt bridges in the folding/unfolding process, (5) to modify deliberately a protein's "non- essential" salt bridges and thereby impart increased stability, (6) to evaluate the role of salt bridges in the stability of proteins in thermophilic organisms. The overall strategy is to relate the conditions of C2N2 treatment ([C2N2], pH, T, t, specific and general salt effects...) of proteins to changes in functionality (enzymatic activity, oxygen binding and cooperativity, pI, polydispersity, functional groups modified...). With these relationships in hand a comparison of the observed cross links with the known salt bridges will be made. The Brookhaven Protein Data Base or Cambridge Data Bank will be used to help identify common features of C2N2 reactive salt bridges. The understanding of the role of structural components is basic to any effort to address structure to function problems and is particularly relevant to current efforts to engineer new protein designs.